miRNAs are endogenous small RNAs (having about 22 nucleotides in sequence), which may specifically bind to genes of particular sequences to regulate the gene expression. At present, numerous studies show that miRNA is associated with the induction of many diseases in human, including cancers, viral infections, cardiovascular diseases, and inflammatory reactions. Control or inhibition of miRNA may provide a strategy for disease treatment. Due to its low toxicity, there have been many treatment strategies with miRNA targeting anti-sense oligonucleotides under clinical trial. The miRNA targeting treatment strategy includes blocking the function of miRNA with some chemically modified anti-sense oligonucleotides, in which the modification is mainly carried out at carbon No. 2 of ribose, for example, replacing the hydroxyl group at carbon No. 2 by 2′-fluoro, 2′-oxy-methyl, 2′-oxyethyl alcohol, 2′-O,4′-C methylene (also referred to as locked nucleic acid (LNA)), 3′-oxyphosphorus sulfide, 2′-deoxyribonucleic acid, and the like. Many anti-sense oligonucleotides successfully enter phase II clinical trial, and have shown to have a safety. For example, the anti-sense oligonucleotide miravirsen (anti-miR122) for treating hepatitis C is a 2′-LNA having 15 nucleotides and 3′-oxyphosphorus sulfide, which can reduce the amount of hepatitis C virus by 100 times in a preclinical trial in chimpanzees, exhibits no adverse effects in phase I clinical trial, and has entered phase II clinical trial (Nature Reviews of Drug Discovery 13, 634, 2014.).
The miRNA targeting anti-sense oligonucleotide therapeutic agent needs to be subjected to bio-distribution and pharmacodynamic test before clinical use. Traditionally, the conventional biodistribution method was done with sacrificed mice, the organs are removed and broken down, and then miRNA is extracted and quantified by enzymatic immunoassay. The major difficulty is the cost. At present, the chemically modified anti-sense oligonucleotide is expensive. It is economic to evaluate the bio-distribution by in-vivo imaging with radioactive label. The metal indium-111 is one of the radioactive isotopes typically used in clinic, which has a medium half-life of 2.8 days and is a good choice in the case of unknown pharmacokinetics of the drug.
Amide bond formation is generally achieved by activating an acid group with an anhydride, an acid ester or with the aid of some conjugating agent such as carbodiimide.
Perreux et al. (Tetrahedron 58(2002), 2155-2162) and Gelens et al (Tetrahedron Letters 2005, 46(21) 3751-3754) report that with the aid of microwave radiation, amide bond formation of benzoic acid with benzylamine is achievable, whereby the amide bonding of 10 mL of the reactant carboxylic acid at mmol level with amine is accomplished, and the yield is increased from the conventional 17% to 80%. However, this has not been used with trace amount of ribonucleic acid, particularly RNA at nmol level. To conjugate a RNA to DTPA, the RNA must have an amino group. Therefore, a long C6-amino arm is terminally added to RNA. The DTPA used is DTPA anhydride or p-isothiocyanate benzyl-DTPA (SCN-Bn-DTPA), such that amide bonding may be carried out under a basic condition. At present, there are few literatures reporting the radioactive label of chemically modified anti-sense oligonucleotide. The DTPA chelating agent generally sequesters indium-111, and used as an imaging agent, to visualize the bio-distribution of the RNA. However, the conventional method suffers from many drawbacks, including long reaction time and low yield.
LNA is known to have the optimum stability among all the chemically modified anti-sense oligonucleotides. There are few studies on imaging of and conjugation of LNA to DTPA. Olivia M. (Bioconjugate Chem. 2009, 20, 174-182) reports that a RNA-DTPA conjugate is successfully formed by reacting 10 nmol RNA in 0.1 M sodium hydroxide (pH 8.0-8.5) with 50 times of p-SCN-Bn-DTPA. However, the yield of the conjugation product is low and is only 32%. Even when the reaction time is extended from 45 min to 3 hrs, the yield of the RNA-DTPA conjugate cannot be increased, because the extended reaction time is unfavorable to the stability of RNA, due to the increased potential of labile breakage. In this regard, attempts are made to react overnight with heating. As analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF), the RNA cannot be completely conjugated to DTPA even more DTPA (50 times) is added. Although the compounds with different molecular weights may be separated by the so-called gel chromatography, the molecular weight of the RNA-DTPA conjugate only slightly differs from that of RNA by about 390-542, and thus the purification is difficult. The disadvantages of long reaction time and incomplete conjugation of RNA to DTPA limit the development of RNA in molecular imaging technology.